PRODUCTION OF BACTERIAL PECTINASE USING BANANA PEELS AGRICULTURAL WASTE AS SUBSTRATE
Amsal Binti Abdul Ghani
QR 82
Bachelor of Science with Honours83 A528 (Resource Biotechnology) 2009 2009
P.KHIDMAT MAKLUMAT AKADeMIK
11111 IIIIrrii 1I1II1I11 1000209938
PRODUCTION OF BACTERIAL PECTINASE USING AGRICULTURAL WASTE AS
SUBSTRATE
Amsal binti Abd. Ghani 15948
This project is submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours (Resource Biotechnology)
Supervisor: Dr. Awang Ahmad Sallehin bin Awang Husaini
Resource Biotechnology Programme Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
2009
Statement of Originality
The work described in this Final Year Project, entitled
"Production of Bacterial Pectinase using Agricultural Waste as Substrate" is to best of the author's knowledge that of the author except where due reference is made
(Date Submitted) Amsal binti Abd Ghani
(15948)
Acknowledgements
First and foremost, I wish to express my deepest and sincerest thanks to my supervisor, Dr.
Awang Ahmad Sallehin bin Awang Husaini who has taught, guided, provided ideas,
supported and encouraged me all the way throughout this study. I am also pleased to thank
all the postgraduate students of Genetic Molecular Laboratory, all staff of Faculty of
Resource Science and Technology and all my colleagues especially to Miss Azeera Aisya
bt. Harnzah for their patient guidance and uncomplaining help during this research . was
carried out. Last but not least, I would like to convey a special gratitude to my parents, Mr.
Abdul Ghani bin Kasa and Mrs. Solhah bt. Haron and also to my siblings, Mrs. Ahlam
Abdul Ghani, Mr. Asyraf Abd. Ghani and Mr. Ajwad Mubarak Abd. Ghani for their love,
advice and constant moral support that has leading me to complete this research.
I
Pusat Khidmat Maldumat Akadelll i . UNTVf'IItSfT' MALAYSIA S WAX
TABLE OF CONTENTS
Acknowledgements
Table of Contents
List ofTables
List of Figures
List ofAbbreviations
Abstract
1.0 Introduction
2.0 Literature Review
2.1 Banana Peels
2.2 Pectin
2.3 Pectinases
2.4 Pectinase-producing Bacteria
2.5 Applications of Bacterial Pectinase
2.6 Production of Pectinase using Agricultural Waste
3.0 Materials and Method
3.1 Initial Isolation of Bacteria
3.2 Enrichment and Serial Dilutions
3.3 Initial Screening
3.4 Screening
3.4.1 Qualitative Screening
3.4.2 Quantitative Screening
3.5 Storage of Culture
I
II
v
VI
VII
1
2
4
4
5
6
7
9
11
14
14
14
14
15
15
16
17
II
3.6 Morphological Characterization 18
3.6.1 Common Morphological Characteristics 18
3.6.2 Gram Staining 18
3.7 Biochemical Characterization 19
3.7.1 Catalase Test 19
3.7.2 Citrate Utilization Test 19
3.7.3 Voges-Proskauer Test 20
3.7.4 Methyl Red Test 20
3.7.5 Hydrogen Sulfate Production Test 20
3.7.6 Motility Test 21
3.8 Molecular Characterization 21
3.8.1 DNA Extraction 21
3.8.2 Agarose Gel Electrophoresis 22
3.8.3 Polymerase Chain Reaction (PCR) 23
3.8.4 Rapid Elution of DNA from Agarose Gels 25
3.8.5 DNA Sequencing 26
3.9 Production ofPectinase 26
3.9.1 Banana Peels 26
3.9.2 Substrate Processing 26
3.9.3 Submerged Fermentation 27
3.10 Optimization Studies for Pectinase Production 27
3.10.1 Effects of Different Percentages of Banana Peels 27
3.10.2 Duration of Fermentation 28
3.10.3 Effects ofDifferent pH 28
3.10.4 Effects ofDifferent Temperature 28
4.0 Results and Discussion 29
4.1 Initial Isolation of Bacteria 29
4.2 Screening 30
4.2.1 Qualitative Screening 30
4.2.2 Quantitative Screening 32
4.3 Gram Staining and Morphological Characterization 34
4.4 Biochemical Characterization 37
4.4.1 Catalase Test 37
III
4.4.2 Citrate Utilization Test 38
394.4.3 Voges-Proskauer Test
404.4.4 Methyl Red Test
414.4.5 Hydrogen Sulfate Production Test
424.4.6 Motility Test
424.5 Molecular Characterization
424.5.1 DNA Extraction
4.5.2 Polymerase Chain Reaction (PCR) 43
4.5.3 Rapid Elution ofDNA from Agarose Gels 45
4.5.4 DNA Sequencing 46
4.6 Pectinase Production using Agricultural Waste as Substrate 47
4.7 Optimization Studies for Pectinase Production 50
4.7.1 Effects of Different Percentages of Banana Peels 50
4.7.2 Duration of Fermentation 53
4.7.3 Effects ofDifferent pH 56
4.7.4 Effects ofDifferent Temperature 59
5.0 Conclusion and Recommendations 61
6.0 References 62
7.0 Appendix 66
IV
J
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 4.1
Table 4.2
Table 4.3
Table 4.4
Table 4.5
Table 4.6
Table 4.7
Table 4.8
LIST OF TABLES
Iodine-potassium iodide solution
DNSA reagent for 1000 ml stock solution
Primer Designation for 16S rONA analysis
PCR reaction mixture
Parameter for PCR Steps
Diameter of halos clearance zones produced by PPB 1 and PPB 2
Polygalacturonase activity of PPB I and PPB 2
Morphological characteristics of PPB 1 and PPB 2 under light microscope and physical observation of the colony on the agar media
Biochemical tests results
Polygalacturonase activity of Bacillus subtilis and Bacillus sp. cp-h24 using 0.25% commercial pectin, 0.25% banana peels and with no pectin as substrates
Polygalacturonase activity ofBacillus subtilis and Bacillus sp. ep-h24 at different duration of fermentation
Polygalacturonase activity ofBacillus subtilis and Bacillus sp. cp-h24 at different pH
Polygalacturonase activity of Bacillus subtilis and Bacillus sp. cp-h24 at different temperatures
v
I J
LIST OF FIGURES
Figure 2.1 Structure of the pectin molecule
Figure 4.1 Halos clearance zone produced by PPB 1
Figure 4.2 Halos clearance zones produced by PPB 2
Figure 4.3 Gram positive of PPB I
Figure 4.4 Gram positive ofPPB 2
Figure 4.5 · Result of Catalase Test ofPPB 1
Figure 4.6 Result of Catalase Test ofPPB 2
Figure 4.7 Result ofVoges Proskauer test
Figure 4.8 Result of Methyl Red Test
Figure 4.9 Result of Hydrogen Sulfide Production Test
Figure 4.10 Agarose gel electrophoresis (1 %) of genomic DNA product from PPB 1 and
PPB. Lanes: 1, PPB 1; 2, PPB 2.
Figure 4.11 Agarose gel electrophoresis (1 %) of PCR products of PPB 1 and PPB 2
using primer pA (forward) and pH (reverse). Lanes: M, 1 kb ladder
(Vivantis); 1, PBB 1; 2, PBB 2.
Figure 4.12 Agarose gel electrophoresis (1 %) of products of rapid elution of DNA from
agarose gel of PPB 1 and PPB 2 that were sent to sequence. Lanes: M, 1 kb
ladder (Vivantis); 1, PPB 1; 2, PPB 2.
Figure 4.13 Polygalacturonase activity ofBacillus subtilis and Bacillus sp. cp-h24 using
0.25 % commercial pectin and banana peels
Figure 4.14 The effects ofdifferent percentages ofbanana peels to the
polygalacturonase activity ofBacillus subtilis and Bacillus sp. cp-h24.
Figure 4.15 Polygalacturonase activity ofBacillus subtilis and Bacillus sp. cp h-24 at
different duration of fermentation.
Figure 4.16. The effects ofpH on the polygalacturonase activity ofBacillus subtilis and
Bacillus sp.cp-h24.
Figure 4.17 The effects ofdifferent temperature to the polygalacturonase activity of Bacillus subtilis and Bacillus sp. cp-h24.
VI
LIST OF ABBREVIATIONS
%w/w
%
°C
III
Ilmol
l6SrDNA
lkb DNA ladder
ANOVA
BLAST
bp
C
CaCh
em
CTAB
dH20
DNSA
dNTPs
EDTA
EtBr
g
giL
H202
M
MgCh
min
ml
mm
mM
MR VP
NaCI
NaOH
weight percent
percent
degree Celcius
microliter
micromol
16S ribosomal DNA
1 kilobase DNA ladder
Analysis of Variance
Basic Local Alignment Search Tool
basepair
carbon
calcium chloride
centimeter
cetyl trimethyl ammonium bromide
distilled water
dinitrosalicylic acid reagents
deoxynucleotide triphosphates
ethylene diamine tetracetic acid
ethidium bromide
gram
. gram per liter
hydrogen peroxide
molar
magnesium chloride
minute
milliliter
millimeter
millimolar
Methyl red-Voges Proskauer
sodium chloride
sodium hydroxide
VII
om
~
peR
PGA
PPB 1
PPB2
!pill
SDS
SIMagar
SmF
SSF
TAEbuffer
TEbuffer
U
UV
V
wlv
YEP medium
a
nanometer
oxygen
polymerase chain reaction
polygalacturonic acid
Pectinase-producing bacteria 1
Pectinase-producing bacteria 2
revolutions per minute
sodium dodecyl sulfate
Sulfate Indole Motility agar
submerged fermentation
solid state fermentation
tris acetate EDTA buffer
tris-EDTA buffer
unit of enzyme
ultraviolet
volt
weight to volume
yeast extract pectin medium
alpha
VIII
Production of Bacterial Pectinase using Banana Peels Agricultural Waste as Substrate
Amsal binti Abdul Ghani
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Bacterial pectinases have emerged as important industrial enzymes with wide-ranging applications recently. III this study, two bacterial strains obtained from orange peels indicated their ability to produce pectinase fiom qualitative and quantitative screening using iodine-potassium iodide and ONS assay method, respectively. Morphological, biochemical and molecular characteristics using 16S rONA analysis were done 10 identify the both strains and their identities are continned to be Bacillus subtilis and Bacillus sp. cp-h24. Production of pectinase from both bacteria strains using banana peels agricultural waste as substrate under aubmerged fermentation has been studied. Higher polygalacturonase activity produced by Bacillus subtilis (13.03 U/m1) and Bacillus sp. cp-h24 (21.18 Vlml) confinned that banana peels can be a better replacement to commercial pectin (9.785 Vlml and 14.14 Vlml, respectively). Optimization studies of the growth medium a wed that Bacillus subtilis produced higher polygalacturonase activity with 10% wlv banana peels (109.8 UIml). at 24 hours fermentation (52.61 Vlml), pH 10 (33.55 Vlml) and temperature 37°C (53.38 Vlml) while IIDciIlus sp. cp-h24 p roduced higher polygalacturonase activity with 5% wlv banana peels (91 .28 Vlml), at 24 hours fermentation (52.33 Vlml), pH 7 (38.27 Vlml) and temperature 37°C (49.66 Vlml). The isolation of pectinase-producing bacteria from agricultural waste and also the use of agricultural waste to produce maximum pectinase suggest strategic ways to reduce the production cost in enzymes commercialization.
Key words: Pectinase(s), Bacillus, production, banana peels agricultural waste
ABSTRAK
Baru-baru ini. pektinase yang diperoleh daripada bakteria telah muncul sebagai enzim yang penting dalam industri dengan mempunyai pelbagai aplikasi. Dalam kajian ini, dua jenis bakteria yang didapati daripada /cuIit oren telah menunjukkan keupayaan dalam menghasilkan pektinase melalui pemilihan secara kualitatif IfIItIIIPnakan lamtan kalium iodida dan melalui pemilihan secara kuantitatif dengan menggunakan kaedah ujilln DNS. Pencirian melalui kriteria moifologi. biokimia dan molekular melalui analisis 16S rDNA telah dl/QIaUr,an untuk mengenal pasti kedua-dua jenis bakteria dan identiti mereka telah dikenal pasti sebagai k jllus subtilis dan Bacillus sp. cp-h24. Penghasilan pektinase daripada kedua-dua bakteria menggunakan Wit pisang sebagai substrat semasa Jermentasi perendaman telah dikaji. Aktiviti poligalakturonase yang leblIt tinggi telah dihasilkan oleh Bacillus subtilis (13.03 Ulml) dan Bacillus sp. cp-h24 (2/.18 Ulm/)) telah lftIIrIbuIctikan lculit p isang boleh menjadi pengganti pektin komersial (9.785 Ulml dan 14.14 Ulml) yang lebih balk. X4jian pengoptimuman media periumbuhan bakteria telah menunjukkan Bacillus subtilis menghasilkan alctiviti poligalakturonase lebih tinggi dengan 10% wlv kulit pisang (109.8 Ulm/). pada 24 jam Jermentasi (52.61 Ulm/). pH 10 (33.55 Ulml) dan suhu 37°C (53.38 Ulml) manakala Bacillus sp. cp-h24 menghasilkan obtvili poligalakturonase lebih tinggi dengan 5% wlv kulit pisang (91.28 Ulml),pada 24 jam Jermentasi (52.33 Ulm/). pH 7(38.27 Ulml) dan suhu 37°C (49.66 Ulml). Pengasingan bakteria yang menghasilkan p«tbuBe daripada baJian buangan dan juga penggunaan bahan buangan. untuk memaksimumkan Jll!lllluDilan pektinase merupakan antara cara yang strategik untuk mengurangkan kos penghasilan dalam
mersialan enzim.
Kala kvnci: Pektinase, Bacillus, penghasilan, kulit pisang
1
,....
1.0 INTRODUCTION
The biotechnological potential of pectinases produced from bacteria has drawn a great deal
of attention for use as biocatalyst in a variety of industrial processes recently. Their a11
embracing applications in degumming and retting of plant fibers, pretreatment of pectic
wastewater from fruit and vegetable processing units (Tewari et al., 2005), degrading the
skin of black pepper to white pepper (Gopinathan and Manilal, 2005), coffee and tea leaf
fennentation, vegetable oil extraction and virus purification (Yadav et al., 2008) have
make these enzymes become the upcoming enzymes of the commercial sector. Besides,
pectinases also have been used in paper and pulp industry, poultry feed additives, alcoholic
beverages and food industries, wood preservation, plant pathology, and in protoplast fusion
technology (Gummadi and Panda, 2003).
Considering the great significance with tremendous potential to offer to industry, one can
conclude that the strategic ways to produce maximum pectinase with low cost production
should be studied. This is because the major impediments to the exploitation of commercial
enzymes are their yield, stability, specificity, and the cost of production. Application of
agricultural waste as carbon sources in enzyme production processes certainly reduces the
cost of production and at the same time helps in preventing environmental pollution (Silva
et al., 2002). Besides, agricultural wastes especially those which contain residues of pectin
are cheap pectinase-producing bacteria providers too. The isolation of pectinase-producing
bacteria from agricultural waste, thus would be one of the another strategic way to
minimize the cost of production.
2
....
Therefore, the objectives of this study are:
1. To screen and isolate pectinase-producing bacteria from pectin residues-containing
agricultural waste.
2. To identify and characterize the isolated pectinase-producing bacteria according to
morphological, biochemical and molecular characteristics.
3. To produce pectinase from the bacteria isolated using banana peels agricultural waste
as substrate under submerged fermentation.
4. To optimize the growth medium of the bacteria isolated for . mwumum pectinase
production.
3
2.0 LITERATURE REVIEW
2.1 Banana Peels
Banana is the general tenn embracing a number of species or hybrids in the genus Musa of
the family Musaceae. In Malaysia, bananas are the second largest cultivated fruit crop after
durian and they are available throughout the year. Generally, they are used as a source of
food in either raw or in processed form.
Bananas are rich in potassium, riboflavin, niacin, and dietary fiber. They also contain
vitamins A and C, B6, some calcium, iron, and magnesium. Because of their sweetness,
they have a high energy value and delicious taste, making them being chosen as one of
preferred source of food for human (Bananas, n.d). This condition actually will lead to the
increased of banana peels and this definitely will contribute to environmental pollution.
One method to solve this problem is to utilize them for the conversion into useful
byproduct. One of valuable byproduct that can be obtained from banana peels is pectin
(Madhavand Pushpalata, 2002).
It has been reported that banana peels contained 0.62% w/w of pectin (Walter, 1991). In
other reports, pectin content in banana peels is 0.7-1.2% w/w (Jayani et ai., 2005). Even
though the amount of pectin in banana peels is not very high compared to pectin from other
fruit wastes such as apple and orange, this amount however is among the highest pectin
content in fruit waste of other local fruit such as papaya, mango and pineapple (Walter,
I). This makes pectin of banana peels are preferable to be utilized as a source of
substrate for pectinases. Stage of banana ripeness also can be contributed to the pectin
4
Pusat Kbidmat Ma tAka. m' UNfVE SIn MALAYSIA ARAWAK
content in banana peels. According to Emaga et al. (2008), stage 5 of banana ripeness
where the colour of banana fruit is more yellow than green has given the greatest yield of
pectin.
2.2 Pectin
Pectin and other pectic substances such as protopectin, pectic acid and pectinic acid are a
group of complex plant polysaccharide that exists mostly in middle lamella and primary
cell wall of plants (Tewari et al., 2005). Hence, they form important natural substrates for
pectinases. They constitute about one third of the cell-wall dry substance of dicotyledonous
and some monocotyledonous plants (Walter, 1991). These substances are believed to
contribute both to the adhesion between cells and to the mechanical strength of the cell,
behaving in the manner of stabilized gels. They also involved in the interaction between
plant hosts and their pathogens (Alkorta et al., 1998).
The principal constituent present in pectin polysaccharides is D-galacturonic acid units
which act as a backbone (Figure 2.1). They joined in chains by means of 0.-(1----+4)
glycosidic linkages. The carboxyl groups of galacturonic acid are partially esterified by
methyl groups and partially or completely neutralized by sodium, potassium or ammonium
ions. Some of the hydroxyl group on C2 and C3 may be acetylated. 2-4% of rhamnose units
are inserted into the main uronide chain and they are joined to the reducing end of the
uronide by (1-2) linkages and the nonreducing end of the next uronide unit by (1----+4)
bonds (Jayani et al., 2005). Often, arabinan, galactan, or arabinogalactan side chains are
ed (1-4) to the rhamnose. In the side chains, the arabinose units have (1----+5) linkages
w e galactoses are mutually joined mainly by (1----+4) linkages, but (1----+3) and (1----+6)
5
linkages also occur. Other sugars, such as o-glucuronic acid, L-fucose, o-glucose, 0
mannose, and o-xylose are also sometimes found in side chains (Walter, 1991).
Figure 1.1 Structure of the pectin molecule. Only one chain of the major component of pectins-galacturonic acid partially methylesterified is represented here. Side chains of galactose, arabinose, xylose and other sugar residues are not included in the figure. Taken from Alkorta et al., (1999).
1.3 Pectinases
Pectinases are a group of heterogenous enzyme that hydrolyzes the pectin and other pectic
substances. The classification of pectinases is based on their attack on the galacturonan
backbone of the pectic substance molecule. Thus, pectinases are broadly classified into
three types. They are de-esterifying enzymes (pectinesterases), depolymerizing enzymes
(hydrolases and lyases) and protopectinases (Alkorta et al., 1998).
Pectinesterases catalyze de-esterification of the methoxyl group of pectin which then
fonning the pectic acid. On the other hand, the depolymerases split the (l (1--+4) glycosidic
bonds between galacturonic monomers in pectic substances either by hydrolysis
(hydrolases) or by ~-elimination (lyases). They have been subdivided into groups by
criteria of whether their preference for pectic acid or pectin as substrate and whether
down starts from within the polymer or from the end of polymer. Enzymes belonged
to lases group are endopolygalacturonase (polygalacturonase), exopolygalacturonase
(polygalacturonase), endopolymethylgalacturonase (pectin hydrolase) and
6
exopolymethylgalacturonase (pectin hydrolase) whereas lyases are endopolygalacturonate
lyase (pectate lyase), exopolygalacturonate lyase (pectate lyase),
endopolymethylgalacturonate lyase (pectin lyase) and exopolymethylgalacturonate lyase
(pectin lyase). Another tyPe of pectinase is protopectinases which is protopectin
solubilizing enzymes that liberate water-soluble and highly polymerized pectin from
protopectin. They are subdivided into A-type and B-type based on preference to react with
polygalacturonic acid region of protopectin (A-type) or with the polysaccharide chains that
connect polygalacturonic acid chain and cell wall constituents (B-type) (Alkorta et al.,
1999).
2.4 Pectinase-producing Bacteria
Pectinases can be produced by a large number ofmicroorganisms such as bacteria (Kapoor
et al., 2000) and fungi (Yadav et al., 2008). Apart from that, some animals (Shen et al.,
1996) and plants (Payasi et al., 2006) are have also been reported to produce this group of
enzyme. Generally, earlier researches focused on fungi such as Aspergillus, Sclerotium,
Penicillium and Rhizopus on their ability to produce pectinases especially Aspergillus niger
because this strain posses GRAS (Generally Regarded As Safe) status, so that metabolites
produced by this strain can be safely used (Gummadi and Panda, 2003). In addition,
pecti.nases from fungi are also widely used because they have acidic pH and this is very
close to the pH of many fruit juices prepared in the juicemaking industry. However, such
pH is not suitable for other preparations in which pH values are close to neutral and alkali.
Furthennore, fungal enzyme has relatively low temperature stability (Soares et al., 1999).
fore, recently researchers are more interested to focus on pectinase from bacteria as a
lot ofpectinase-producing bacteria have been successfully isolated included Pseudomonas,
7
other
Aspergillus niger and
Bacillus, Erwinia and Clostridium. In contrast to fungi, bacteria like Bacillus licheniformis
are predominately produced alkaline pectinases and were thennostable because of their
ability to retain 100% activity after 2 hours of incubation at 65°C used to have been
reported (Singh et al., 1999). Besides, a novel, alkaline and thennostable polygalacturonase
has been produced from an envirorunental isolate Bacillus sp. MG-cp-2 in which that
polygalacturonase is not only active, stable under highly alkaline conditions and has broad
l'8Ilge of pH (7.0-12.0), but also exhibits a broad range of thennostability at temperatures
up to 80°C, also tolerance to metal ions and surface-active agents (Kapoor et al., 2000).
Smmotel and Nigam (2002) have successfully examined the production of different
peetinolytic activities in five alkalophilic soil bacteria under submerged fennentation by
using pectin as substrate. The result has shown that maximal activity of alkaline pectin
lyase and polygalacturonase have been produced by all the five bacteria. In addition, both
activities were higher than most other microorganisms reported in literature. Kashyap et al.
(2000) also has reported that Bacillus sp. DT7 has produced higher yield of pectinase
compared to most other microbes such as Aspergillus and Penicillium. Better yield along
with the advantages of its alkalophilic and thennotolerant properties have indicated that
this organism has the pot~tial to be used at commercial level for degumming of natural
fibers, in pretreatment of waste water from fruit-juice processing industry and also many
industrial applications. Soares et al. (1999) also reported that activities of
lygalacturonase produced by Bacillus sp. strains were higher than those produced by
Tubercularia vulgaris. The other bacteria that has the same
properties and has been successfully isolated is Bacillus sp. KSM-P443 (Koboyashi et al.,
8
•
In a lot more other reports, bacteria have been admitted and justified as one of the great
producer of pectinases. For example, Bacillus subtilis IFO 12113 (Sakai and Ozaki, 1988)
and Bacillus subtilis IFO 3134 (Sakai and Sakamoto, 1990) have been reported to produce
B-type protopectinase. On the other hands, pectate lyase have been reported to be
produced by a wide range of bacteria such as Bacteroides thetaiotaomicron (McCarthy et
aL, 1985), Erwinia carotovora (Kotoujansky, 1987), Amycolata sp. (Bruhlmann, 1995)
Pseudomonas syringae pv. Glycinea (Margo et al., 1994), and Erwinia chrysanthemi
(Favey et al., 1992). Pectinesterase also have been reported to be produced by bacteria
such as Erwinia chrysanthemi B341 (Pitkanen et al., 1992), Lachnospira pectinoschiza
(Cornick et al., 1994), Pseudomonas solanacearum (Schell et al., 1994) and Lactobacillus
Iactis subsp. Cremoris (Karam and Belarbi, 1995).
2.5 Applications of Bacterial Pectinase
Bacterial pectinases have great importance in industrial processes such as degumming and
retting of natural fibers (Kashyap et al., 2000). The application of pectinase in these
processes not only reduces the overall cost, but also helps to conserve energy and preserve
the environment (Kapoor et al., 2000) as the chemical retting and degumming treatment is
polluting, toxic and non-biodegradable. Retting and degumming process using pectinases
presents an envirorunental friendly and economic alternative to the problem (Jayani et al.,
20(5).
In retting process, pectinases have been used in retting of flax to separate the fibers and
e1bnjnate the pectins since pectinases are believed to playa lead role in fiber processing,
40% of the dryweight of plant cambium being pectin (Kapoor et al., 2000). Yadavet
20(8) have been reported that pectin lyases produced from Aspergillus jlavus have the
9
.
efficient degumming
ability in retting the Croto/aria juncea sticks or known as sunn hemp only in 24 hours.
lDtetestingly, the retting process is done with the absence of EDT A, a chemical which
usually use to enhance the retting efficiency.
In degmulJling process, pectinases are usual1y used in degumming of plant bast fibers
(Jayani et al., 2005). The fibers contain gummy materials consisting mainly of pectin and
hemicelluloses which have to be removed before being use in textile making (Kapoor et
tIL. 2(01). A study by Kapoor et al. (2001) has shown that alkaline and thermostable
polyplacturonase produced from Bacillus MG-cp-2 has the ability in degumming of
BoeIrmeria nivea (ramie) and Crotolariajuncea (sunn hemp) bast fibers by 37% and 56%
respectively only in 11 hours with the help of a few chemicals. The process of degumming
enhanced by its stability in high temperature and pH, which is the pre-requisite for an
process. Besides, culture supernatants contained pectate lyase
produced from Amycolata sp. has also been reported to effectively reduce the gum content
ramie fibers by 30% only in 15 hours (Bruhlmann et al., 2000).
One other appreciative application of bacterial pectinase that also should be given more
atteDtiOD is the potential of pectinase in reducing the time needed to degrade the skin of
pepper and green pepper to white pepper. Considering that white pepper is more
Crred than black or greeD pepper in some country such as Europe, Japan and America
1;JJaII:IWIC of its channing colour, suitability to use in all food preparations, more starch
I,COlIltaiDing, less microbial load and contaminant-free (Gopinathan and Manilal, 2005), it
to conclude that white pepper has a very high commercial value. Thus, the great
ofpectinase regarding this matter should not be ignored.
10
(Revilla
Pectinases also has the potential to be applied in more other applications such as in waste
water treatment and in coffee and tea fermentation. Vegetable food processing industries
release pectin, containing waste waters as by-products. Pretreatment of these wastewaters
with pectinolytic enzymes facilitates removal of pectinaceous material and renders it
'table for decomposition by activated sludge treatment (Boondal et al., 2000). In coffee
and tea fennentation on the other hands, pectinase treatment accelerates tea fermentation
and also destroys the foam forming property of instant tea powders by destroying pectins
(Carr, 1985), They are also used in coffee fermentation to remove mucilaginous coat from
coffee beans.
Pechna se8 also are believed can help in improvement of chromaticity and stability of red
wines in alcoholic beverages industry. Pectinases added to macerated fruits before the
addition of wine yeast in the process of producing red wine resulted in improved visual
characteristic (colour and turbidity) as compared to the untreated wines. Enzymatically
treated red wines presented chromatic characteristics, which are considered better than the
aoatrol wines, These wines also showed greater stability as compared to the control
and Ganzalez-san jose, 2003). In paper making industry, pectinase can
depolymerize pectins and subsequently lower the cationic demand of pectin solutions and
the filtrate from peroxide bleaching (Viikari et al., 2001).
Production of Pectinase using Agricultural Waste
BYildel!1ce showed that pectinases are inducible and they can be produced with the presence
1'~lIifti:relllt carbon sources (Aguilar and Buitron, 1987; Maldonado et al., 1989; Friedrich
111.. 1994; Nair et al., 1995; Nair and Panda, 1997). In the course of time, numerous
11
RlPD11S
O
have appeared on the different strategies for maximizing the production of
ped:inases with the reduction of production cost. Soares et al., (1999) reported on
production ofpolygalacturonase from Bacillus sp. under solid state fermentation (SSF) and
submerged fennentation (SmF) by using wheat bran as the substrate and the results shown
that the wheat bran can be used as a good substrate replacing the commercial pectin.
Kashyap et al., (2002) also has enhanced the production of pectinase by Bacillus sp. DT7
by using a few types of natural solid substrates such as wheat bran, rice bran and apple
pomace under SSF. His report also shows the same results with Soares et al., (1999) and
the wheat bran has been the prime producer among all the substrates that have been used.
Furthermore, Kapoor et al., (2000) also has revealed that complex polysaccharides such as
agricultural wastes and agro-industrial by-products are the better stimulant for the
polygalacturonase production from Bacillus sp. MG cp-2 which has the properties of
thermo-alkali stable. The use of wheat bran, sunflower seed cake, rice bran, orange peel
IDd guar gum was enhanced the polygalacturonase production to a significant extent when
substituted individually in place of commercial citrus pectin. On the other hands, Bayoumi
et alo, (2008) used potato peels for cost-effective production of the pectinase. Solanum
tuberosum peels · and Solanum melanogena peels have been used as the substrates to
produce pectinase from Bacillus firmus under solid state fermentation and those cheap
substrates have shown to be the great substrates.
des that, Silva et al., (2002) have produced pectinase from Penicillium Viridicatum
under solid state fennentation using many types of agricultural wastes and agro
IDdllStlrial by-products such as orange bagasse, sugar cane bagasse, wheat bran, banana and
1(81Il10 peels and com teguments. The results showed that all those agricultural wastes and
12
by-products are suitable to be used . as carbon source for Penicillium
WllIlfdiaa-. as they produce polygalacturonase and pectin lyase after 2 to 12 days of
l3